Composition having lactobacillus plantarum strain gmnl-662 for promoting bone regrowth

ABSTRACT

A composition having  Lactobacillus Plantarum  strain GMNL-662 for promoting bone regrowth is provided. The  Lactobacillus Plantarum  strain GMNL-662 has an ability to promote the expression of osteogenic genes, inhibit the expression of osteoclast related genes, and promote the expression of osteogenesis-related cytokine TGF-β, so that the bone loss is improved.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the filing date of Taiwan patentapplication No. 106105625, filed on Feb. 20, 2017, the disclosure ofwhich is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to a composition including LactobacillusPlantarum strain GMNL-662 for promoting bone regrowth, and in particularrelates to a composition including Lactobacillus Plantarum strainGMNL-662 which has an ability of increasing the expression of osteogenicgenes.

BACKGROUND OF THE INVENTION

Osteoporosis is a kind of systemic skeletal disease, which includes boneloss and bone tissue microstructure deterioration, resulting in bonefragility and risk of fracture.

During bone remodeling process, the bone formation of osteoblasts andbone resorption of osteoclast maintain the dynamic balance of bonetissue together. Once the bone resorption is over bone formation, boneloss will caused, and finally result in osteoporosis. In general,osteoporosis can be divided into postmenopausal osteoporosis and senileosteoporosis. Postmenopausal osteoporosis is common in women aftermenopause, due to the rapid reduction of estrogen in the female body, sothat the osteoclast activity is increased to absorb the trabecular bone,and ultimately make the trabecular bone thinning, broken off, and makethe number of the bone cells reduced or discontinuous, resulting inreduction of bone strength. Senile osteoporosis is caused by the declineof osteogenic cell function, insufficient calcium and vitamin D intake,intestinal absorption dysfunction, leading to reduced bone synthesis,thick loose cortical bone, and trabecular bone disappeared, so that bonestrength is significantly reduced.

According to its mechanism, the current drugs for prevention andtreatment of osteoporosis and fracture can be divided intoanti-osteoclast or anti-loss drugs, bone formation or promotingosteoblast drugs, and mixed type drugs. Anti-osteoclast drugs includecalcium, vitamin D, calcitonin, bisphosphonates, estrogen receptormodulators, sex hormones, osteoclast enzyme inhibitors, RANKL monoclonalantibody. The mixed type drug is currently strontium salt only. Thedrugs that control osteoporosis are accompanied by some side effects. Itis found in the clinical trials that the use of drugs in combination hasno addition effect, but will resist each other, or increase theincidence or strength of the side effects. Therefore, the currentguidelines for various prevention and treatment of osteoporosis are notrecommended to use two anti-loss reagents, or use one anti-loss reagenttogether with one promoting osteoblast reagent.

The osteoporotic drugs clinically used in the elderly and menopausalwomen, such as Fosamax, Tevanate, Covaxin (bisphosphonates drugs), willcause serious necrosis of jaw bone joint if users do not pay attentionto oral hygiene, or the users are subject to tooth extraction, dentalimplant surgery. Recent studies have also found that it may cause theadverse reactions including atypical femoral fracture.

Although some literatures state that certain specific probiotic strains,for example: L. reuteri ATCC PTA 6475; L. paracasei DSM13434; L.plantarum DSM 15312, DSM 15313 and B. longum, have the ability to reducebone loss in ovariectomized rats, but they are applied in the form oflive bacteria in the experiments, and it is found that the ability toslow down bone loss is achieved by reducing inflammation. Theabovementioned strains do not have the ability to make boneregeneration, and thus the treatments are more passive.

It is therefore necessary to provide a composition for promoting boneregrowth, in order to solve the problems existing in the conventionaltechnology as described above.

SUMMARY OF THE INVENTION

A primary object of the present invention is to provide a compositioncomprising Lactobacillus Plantarum strain GMNL-662 for promoting boneregrowth. The Lactobacillus Plantarum strain GMNL-662 can beadministrated through any possible pathway in order to enter thedigestive system to increase the gene expression of osteogenesis-relatedcytokine TGF-β and osteocalcin, and inhibit the expression of osteoclastrelated genes (such as TRAP-5), thereby solving the problem caused bybone loss.

To achieve the above objects, the present invention provides acomposition for promoting bone regrowth, comprising LactobacillusPlantarum strain GMNL-662 deposited in the China Center for Type CultureCollection (CCTCC) with an accession number of CCTCC M 2016571.

In one embodiment of the present invention, the Lactobacillus Plantarumstrain GMNL-662 is a viable strain or a dead strain.

In one embodiment of the present invention, the Lactobacillus Plantarumstrain GMNL-662 has an ability to improve the expression of osteogenicgenes.

In one embodiment of the present invention, the osteogenic genescomprise osteocalcin gene.

In one embodiment of the present invention, the composition is apharmaceutical composition, a nutritional supplement, a health food, amedical food, or the combination thereof.

In one embodiment of the present invention, the composition is appliedto slow down bone loss.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing the expression of cytokine TGF-β of eachgroup in the experiment 2 according to one embodiment of the presentinvention.

FIG. 2 is a diagram showing the expression of osteogenesis-related geneosteocalcin of each group in the experiment 2 according to oneembodiment of the present invention.

FIG. 3 is a diagram showing the expression of osteoclast related geneTRAP-5 of each group in the experiment 2 according to one embodiment ofthe present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The structure and the technical means adopted by the present inventionto achieve the above and other objects can be best understood byreferring to the following detailed description of the preferredembodiments. Furthermore, if there is no specific description in theinvention, singular terms such as “a”, “one”, and “the” include theplural number. For example, “a compound” or “at least one compound” mayinclude a plurality of compounds, and the mixtures thereof. If there isno specific description in the invention, “%” means “weight percentage(wt %)”, and the numerical range (e.g. 10%-11% of A) contains the upperand lower limit (i.e. 10%≤A≤11%). If the lower limit is not defined inthe range (e.g. less than, or below 0.2% of B), it means that the lowerlimit may be 0 (i.e. 0%≤B≤0.2%). The proportion of “weight percent” ofeach component can be replaced by the proportion of “weight portion”thereof. The abovementioned terms are used to describe and understandthe present invention, but the present invention is not limited thereto.

One embodiment of the present invention provides a LactobacillusPlantarum strain for promoting bone regrowth. The LactobacillusPlantarum strain is referred to as Lactobacillus Plantarum strainGMNL-662, which is deposited in the China Center for Type CultureCollection (CCTCC) with an accession number of CCTCC M2016571.

One embodiment of the present invention provides a composition forpromoting bone regrowth, comprising the abovementioned LactobacillusPlantarum strain GMNL-662. Preferably, the composition can be apharmaceutical composition, a nutritional supplement, a health food, amedical food, or the combination thereof. The composition can be formedin various form based on the effectively or convenience. In addition,the composition is preferably administrated by means of food to enterthe digestive system, and can stimulate the expression of osteogenicgenes, inhibiting the expression of osteoclast related genes, andpromoting the expression of osteogenesis-related cytokine TGF-β to slowdown bone loss.

The Lactobacillus Plantarum strain GMNL-662 in the abovementionedembodiments is one of a plurality of isolates mainly isolated from humanintestines. The primers (SEQ ID NO: 1 and SEQ ID NO: 2) listed in Table1 are used to perform PCR to reproduce 16S rDNA segments of eachisolate, and then sequencing the 16S rDNA segment of each isolate. Aftersequencing, a 16S rDNA gene sequence of one of the isolates can beobtained as below (SEQ ID NO: 3); subsequently, from the comparisonresults on the NCBI website, it shows that the 16S rDNA sequences of theisolates are similar to that of the Lactobacillus Plantarum strains withidentities all over 99%, so that the strain GMNL-662 indeed belongs tothe Lactobacillus Plantarum genus.

TABLE 1 PCR primer SEQ Primer ID NO: SEQ PAF 1AGA GTT TGA TCC TGG CTC AG 536R 2 GTA TTA CCG CGG CTG CTG

TABLE 2 NCBI NO Description Identity KT236093.1 Lactobacillus plantarumKLB 410 99% KT962240.1 Lactobacillus plantarum USIM03 99% KT025848.1Lactobacillus plantarum KF 99% KR818164.1 Lactobacillus plantarum KF999%

A complete 16S rDNA sequence (SEQ ID NO: 3) of the LactobacillusPlantarum strain GMNL-662 is listed as below:

GCCGTTGGCGTCGGATACATGCATGTCGTACGAACTCTGGTATTGATTGGTGCTTGCATCATGATTTACATTTGAGTGAGTGGCGAACTGGTGAGTAACACGTGGGAAACCTGCCCAGAAGCGGGGGATAACACCTGGAAACAGATGCTAATACCGCATAACAACTTGGACCGCATGGTCCGAGCTTGAAAGATGGCTTCGGCTATCACTTTTGGATGGTCCCGCGGCGTATTAGCTAGATGGTGGGGTAACGGCTCACCATGGCAATGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAACTCTGTTGTTAAAGAAGAACATATCTGAGAGTAACTGTTCAGGTATTGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGGTAAACAC

A fermentation test to the Lactobacillus Plantarum strain GMNL-662 iscarried out to obtain the results shown in Table 3.

TABLE 3 Fermentation Test Strips No. carbohydrates substrate GMNL-662 0CONTROL − 1 Glycerol − 2 Erythritol − 3 D-Arabinose − 4 L-Arabinose − 5D-Ribose + 6 D-Xylose − 7 L-Xylose − 8 D-Adonitol − 9Methyl-β-D-Xylopyranoside − 10 D-Galactose + 11 D-Glucose + 12D-Fructose + 13 D-Mannose + 14 L-Sorbose − 15 L-Rhamnose − 16 Dulcitol −17 Inositol − 18 D-Mannitol + 19 D-Sorbitol + 20Methyl-α-D-mannopyranoside + 21 Methyl-α-D-glucopyranoside − 22 N-Acetylglucosamine + 23 Amygdalin + 24 Arbutin + 25 Esculin ferric citrate − 26Salicin + 27 D-Cellobiose + 28 D-Maltose + 29 D-Lactose (bovineorigin) + 30 D-Melibiose + 31 D-Saccharose (sucrose) + 32 D-Trehalose +33 Inulin − 34 D-Melezitose + 35 D-Raffinose − 36 Amidon (starch) − 37Glycogen − 38 Xylitol − 39 Gentiobiose + 40 D-Turanose + 41 D-Lyxose −42 D-Tagatose − 43 D-Fucose − 44 L-Fucose − 45 D-Arabitol − 46L-Arabitol − 47 Potassium gluconate + 48 Potassium 2-ketogluconate − 49Potassium 5-ketogluconate − −: negative; +: positive

To verify the bone regrowth properties of the Lactobacillus PlantarumGMNL-662 according to the present invention, and to confirm that thebone loss can be improved, experiments 1 to 3 are executed.

Experiment 1: Bone Tissue Analysis

Strain: Lactobacillus plantarum Strain GMNL-662

Strain Treatment:

(1) preparation of viable bacteria: Inoculating the LactobacillusFermentum GMNL-296 from a frozen tube to 1 ml of MRS broth, and standingunder 37° C. for aerobically incubating for 20 hours. The next day,adding 15 μl culture solution into 1.5 ml of MRS broth (1% secondaryactivation), and then standing under 37° C. for aerobically incubatingfor 20 hours. Estimating the bacteria number by using OD 600 to adjustthe bacteria concentration to 8×10⁷ CFU/ml.

(2) preparation of dead bacteria: Inoculating the LactobacillusPlantarum strain GMNL-662 from a frozen tube to 1 ml of MRS broth, andstanding under 37° C. for aerobically incubating for 20 hours. The nextday, adding 15 μl culture solution into 1.5 ml of MRS broth (1%secondary activation), and then standing under 37° C. for aerobicallyincubating for 20 hours. Estimating the bacteria number by using OD 600to adjust the bacteria concentration to 4.1×10⁸ CFU/ml.

Osteoporosis Mouse Model:

8-week-old ICR female rats were purchased from BioLASCO Taiwan andovariectomy was performed when they were 9 week-old. Mice were underwentanesthesia and were ovariectomized through back on both sides of theovaries. All groups were given the test substance by means of tube feedat 4 days after surgery. The groups were divided into a sham operationgroup (control group, their abdominal cavity were cut but their ovarieswere not removed); and 4 groups ovariectomized group (Ovariectomy; OVX).When the mice were sacrificed, the ovarian tissues were checked andconfirmed whether the removal of ovarian was successful. Theexperimental results of the mice under failure operation were not used.In the 4 groups of the ovariectomized mice, one group was the vehiclegroup (H₂O group), and one group was the positive drug group(anti-osteoporosis drug Alendronate). Alendronate was formulated withdeionized water at a concentration of 0.25 mg/ml. The mice were given0.1 ml per 10 grams of body weight and 4 times a week. The remaining twogroups were fed with 0.2 ml of alive GMNL-662 (strain concentration is8×10⁷ cfu/ml; daily dose of the mouse is 1.6×10⁷ cfu/mouse, the humandose is 4×10⁹ cfu/60 kg adult), and 0.2 ml of dead GMNL-662 (strainconcentration is 4.1×10⁸ cfu/ml; daily dose of the mouse is 8.2×10⁷cells/mouse, the human dose is 2×10¹⁰ cells/60 kg adult). The two groupswere fed with tube one time every day, continued for 28 days, the micewere anesthetized and sacrificed for intraperitoneal cephalic veinsampling, and each femur was removed for analysis.

Analysis Method:

The backbone of the right femur far from the end was taken a computertomography by a micro computed tomography (SkyScan 1076, Kontizh,Belgium, with resolution of 18 μm), and the trabecular bone volume ratio(i.e. bone volume/l tissue volume) is analyzed by a software. Theanalyzed position was selected to include the area of 100 pieces underthe growth plate excluding cortical bone. The bone mineral densityanalysis was applied to the same area. The obtained data in theexperiments were analyzed with two-way analysis of variance, andexecuted T-test statistical analysis. All data were presented asmean±SD. After comparisons, the abovementioned groups were analyzedstatistically and noted by different marks to represent thestatistically significant differences (* represents p<0.05: **represents p<0.01). See Table 4 and Table 5, showing results of theexperiment 1.

TABLE 4 Trabecular bone volume ratio (BV/TV, bone volume/tissue volume)BV/TV (%) OVX + OVX + OVX + GMNL-662 GMNL-662 OVX + Control H₂O alivedead Alendronate 42.12 ± 30.9 ± 36.92 ± 36.8 ± 34.88 ± 2.4** 1.1 1.7**1.2** 0.9**

From table 4, after removing the ovarian, the trabecular bone volumeratio in (OVX+H₂O) group (disease group) was lower than the controlgroup, which means that the osteoporosis animal model was successful.Comparing the alive GMNL-662 group with the dead GMNL-662 group, it canbe found that BV/TV thereof were higher than the disease group, whichmeans that the GMNL-662 indeed slows down bone loss in a certain degreeafter removing the ovarian. Alendronate was positive control group whichalso had protective effect against bone loss. The two groups of the tubefed GMNL-662 strains even have slightly better protective effects thanthe anti-osteoporosis drug Alendronate.

TABLE 5 Femur bone mineral density (BMD, excluding cortical bone) BMD(g/cm³) OVX + OVX + OVX + GMNL-662 GMNL-662 OVX + Control H₂O Alive DeadAlendronate 0.502 ± 0.344 ± 0.488 ± 0.474 ± 0.426 ± 0.04** 0.04 0.02**0.01** 0.02*

From table 5, it can be noted that the disease group (OVX+H₂O) has lowerBMD than the control group; in the groups of alive and dead GMNL-662strains, the BMD is significantly higher than the BMD in the diseasegroup (OVX+H₂O). That is, both of the two groups of the tube fedGMNL-662 strains can slow down bone loss of the mice after removing theovarian.

Experiment 2: Effects of GMNL-662 on Osteogenic Genes, Cytokines, andOsteoclast Genes

Extraction of tibial RNA: The left femur of the mice were removed, cutinto small pieces with scissor, and an appropriate amount of liquidnitrogen was added to grind the bones quickly. The ground bone powderwas added to 0.5 ml TRIzol® Reagent to extract RNA; 0.1 ml chloroformwas then added thereto to turn up and down 15 times. The solution wasplaced at room temperature to react for 5 minutes, followed bycentrifugalized and extracted the upper layer to new microcentrifugetubes (eppendorf); 0.25 ml isopropanol was added thereto and thesolution was placed at room temperature for 10 minutes and thencentrifugalized; the supernatant was removed and the precipitate waswashed with 0.5 ml 75% ethanol; after the precipitate was dried, 20-50μl DEPC water was added to dissolve the precipitate and the RNAconcentration was measured.

RNA reverse transcription cDNA: 1-5 μg RNA was obtained and RNase-freewater was added therein to 10 μl; additionally, 10× Random primer (2μl), 10 mM dNTP (1 μl) were added, at 65° C. for 5 minutes, and on icefor 2-3 minutes; after first stage interaction, additional 5×RT buffer(4 μl), 0.1M DTT (1 μl), RNase inhibitor (Invitrogen, RNaseOUT™, 1 μl),RT enzyme (Invitrogen, SuperScript® III, 1 μl) were added and mixed atroom temperature for 5 minutes, and then placed at 50° C. for 60minutes, at 70° C. for 15 minutes, to proceed the enzyme reversetranscription.

Tibial cDNA in real-time PCR analysis: 1 μl tibial cDNA was obtained andadded 4 μl of 1 μM F+R primers (forward/reverse primers are listedbelow), and 5 μl of 2× Rotor-Gene SYBR Green PCR Master Mix (Qiagen,Cat. 204076), placed into Q-PCR apparatus to react. The relativeexpression of TGF-β and RANKL were obtained by deducting the GAPDHitself.

TABLE 6 Primers TGF-β Forward SEQ ID NO: 4 GAGTAACGCTTTCCG primer GAGTCTGF-β Reverse SEQ ID NO: 5 ACAGTCACCAGCATC primer TCAGC OsteocalcinSEQ ID NO: 6 ACGGTATCACTATTT Forward primer AGGACCTGTG OsteocalcinSEQ ID NO: 7 ACTTTATTTTGGAGC Reverse primer TGCTGTGAC TRAP-5 ForwardSEQ ID NO: 8 GACGATGGGCGCTGA primer CTTCA TRAP-5 Reverse SEQ ID NO: 9GCGCTTGGAGATCTT primer AGAGT GAPDH Forward SEQ ID NO: 10 GCACAGTCAAGGCCGprimer AGAAT GAPDH Reverse SEQ ID NO: 11 GCCTTCTCCATGGTG primer GTGAA

Analysis method: The obtained data in the experiments were analyzed withtwo-way analysis of variance, and executed T-test statistical analysis.The abovementioned groups were analyzed statistically compared with theOVX+H₂O group, wherein * represents p<0.05; ** represents p<0.01.

As shown in FIG. 1, GMNL-662 alive strain and dead strain both canincrease the expression of cytokine TGF-β which can protect bone againstbone loss. Comparing the mice of the control group with the diseasegroup (OVX+H₂O), the expression of bone regrowth related cytokine TGF-βof the disease group is significantly reduced; the mice fed withGMNL-662 (GMNL-662 alive strain and dead strain) have significantincreased expression of TGF-β compared with the disease group (OVX+H₂O).This result means that the GMNL-662 strain has ability of promoting theexpression of TGF-β so as to slow down bone loss.

Next, as shown in FIG. 2, in the groups of the mice given GMNL-662(alive or dead strain) after removing ovarian, the expression ofosteocalcin gene are higher than the disease group (OVX+H₂O), whichmeans that the GMNL-662 strains, no matter the strain is dead or alive,have ability to promote the expression of osteogenic genes, especiallyosteocalcin gene, thereby slowing down bone loss.

Refer to FIG. 3, it can be observed apparently, compared with the micein the sham operation group (Control), the expression of osteoclastrelated genes TRAP-5 in the disease group (OVX+H₂O) is increased; whilein the groups of GMNL-662 alive strain and dead strain, the expressionof osteoclast related genes TRAP-5 is significantly lower than that inthe disease group (OVX+H₂O). That is, GMNL-662 strains have ability toinhibit the expression of osteoclast genes so as to slow down bone loss.

In summary, according to the above results, it is certain that theLactobacillus Plantarum strain GMNL-662 according to the presentinvention, no matter the stains are viable or dead, can significantlyslow down bone loss of the mice after removing ovarian in the bonetissue analysis (Trabecular bone volume ratio, BV/TV) of animalexperiments and bone mineral density (BMD). It is also noted that theGMNL-662 strain has abilities of promoting the expression of osteogenicgenes (Osteocalcin gene), inhibiting the expression of osteoclastrelated genes (TRAP-5), and promoting the expression ofosteogenesis-related cytokine TGF-β, so that the bone loss can beimproved. In addition, it can be found in the experiment results thatthe GMNL-662 has protective effect better than the anti-osteoporosisdrug “Alendronate”. Alendronate has been found to have many sideeffects, including heart disease, stubborn pain, jaw osteonecrosis,fractures, and esophageal cancer. Therefore, Lactobacillus Plantarumstrain GMNL-662, safe and with no side effects, is applicable to slowdown bone loss. It should be a better choice for the menopausal women inconsidering the future prevention and improvement of bone loss.

The present invention has been described with preferred embodimentsthereof and it is understood that many changes and modifications to thedescribed embodiments can be carried out without departing from thescope and the spirit of the invention that is intended to be limitedonly by the appended claims.

What is claimed is:
 1. A composition for promoting bone regrowth,comprising Lactobacillus Plantarum strain GMNL-662 deposited in theChina Center for Type Culture Collection (CCTCC) with an accessionnumber of CCTCC M2016571.
 2. The composition according to claim 1,wherein the Lactobacillus Plantarum strain GMNL-662 is a viable strainor a dead strain.
 3. The composition according to claim 1, wherein theLactobacillus Plantarum strain GMNL-662 has an ability to improve theexpression of osteogenic genes.
 4. The composition according to claim 3,wherein the osteogenic genes comprise osteocalcin gene.
 5. Thecomposition according to claim 1, wherein the composition is apharmaceutical composition, a nutritional supplement, a health food, amedical food, or the combination thereof.
 6. The composition accordingto claim 1, wherein the composition is applied to slow down bone loss.